This project aims to identify novel small molecules inhibitors of DHS and DOHH that may prove to be useful anticancer agents. During this period, the collaborative team has successfully developed a 384-well high-throughput DHS assay which enables the direct detection of enzymatic products Rapidfire mass spectrometry. Assay conditions and parameters such as incubation time, Km, Z' and the signal-to-background ratio have been optimized and determined to be sufficiently robust to support high-throughput screening against NCGC's small molecule collections.